AbstractSerological classification of individuals as A, B, O, or AB is a mainstay of blood banking. ABO blood groups or ABH antigens, in addition to other surface glycans, act as unique red blood cell (RBC) signatures and direct immune responses. ABO subgroups present as weakened, mixed field, or unexpected reactivity with serological reagents, but specific designations remain complex. Lectins detect glycan motifs with some recognizing ABH antigens. We evaluated a 45-probe lectin microarray to rapidly analyze ABO blood groups and associated unique glycan signatures within complex biological samples on RBC surface glycoproteins. RBC membrane glycoproteins were prepared from donor RBCs, n = 20 for each blood group. ABO blood group was distinguishable by lectin array, including variations in ABH antigen expression not observed with serology. Principal component analysis highlighted broad ABO blood group clusters with unexpected high and low antigen expression and variations were confirmed with ABH antibody immunoblotting. Using a subset of lectins provided an accurate method to predict an ABO serological phenotype. Lectin microarray highlighted the importance of ABO localization on glycoproteins and glycolipids and pointed to increased glycocalyx complexity associated with the expression of A and B antigens including high mannose and branched polylactosamine. Thus, lectins identified subtle surface ABO blood group glycoprotein density variations not detected by routine serological methods. Transfusion services observe alterations in ABH expression during malignancy, and ABO incompatible solid organ transplantation is not without risk of rejection. The presented methods may identify subtle but clinically significant ABO blood group differences for transfusion and transplantation.